2.10. Western Blotting
Briefly, equal amounts of total cell lysates were blotted onto polyvinylidene fluoride membranes, incubated with primary antibodies overnight at 4°C, washed, and incubated with peroxidase-conjugated secondary antibody for 1 h at 37°C. The primary antibodies against p-RIP1 (#65746), RIP1 (#33493), p-MLKL (#37333), MLKL (#14993), and SOD-2 (#13194s) were obtained from Cell Signaling Technology (Danvers, MA, USA). RIP3 (ab62344), GCLC (ab53179), Foxo3 (ab47285), GCLM (ab153967), HO1 (ab68477), and NQO1 (ab80588) were obtained from Abcam (Cambridge, MA, USA). The β-actin antibody (sc-47778) and peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG were obtained from ZSGB-BIO (Beijing, China). Immuno-complexes were visualized on a luminescent imaging workstation (6600; Tanon, Shanghai, China), and electrochemiluminescence was detected with BeyoECL Plus (Beyotime Institute of Biotechnology, Beijing, China). Proteins were quantified using scanning densitometry with ImageJ software (National Institutes of Health, USA). Three independent experiments were performed.